Chromogenic merocyanine enzyme substrates

ABSTRACT

Chromogenic merocyanine enzyme substrate compounds of the general formula: ##STR1## where Y is an enzymatically-cleavable group such as a radical of a sugar, carboxylic acid, amino acid, peptide, phosphoric acid, or sulfuric acid; A and B represent residues that complete 5- or 6-membered ring systems; R 1  is substituted or unsubstituted alkyl; R 2  and R 3 , independently, are hydrogen or lower alkyl; m, n, and p, which can be different, are integers from 0 through 3 provided that m+n+p must be at least 2; and X is an appropriate counterion (anion).

This is a division of application Ser. No. 310,060, filed on Feb. 13,1989, now U.S. Pat. No. 5,122,602.

BACKGROUND OF THE INVENTION

The present invention relates to chromogenic compounds which are usefulas optical indicator compounds in analytical test systems. Inparticular, the present invention relates to novel chromogenic enzymesubstrate compounds and their use in analytical test systems for thedetection of enzymes in a liquid test sample.

The determination of enzymes is important in a variety of fields such asbiochemical research, environmental and industrial testing, and medicaldiagnostics. The quantitation of enzyme levels in body fluids such asserum and plasma provides very useful information to the physician indiagnosing diseased states and their treatment. In addition to beinganalytes of interest in biological fluids, enzymes can also serve asdetection reagents in a variety of analytical systems such asimmunoassays and nucleic acid hybridization techniques. In such systems,enzymes are useful directly or indirectly as labels to monitor theextent of antigen-antibody binding or nucleic acid hybridization thatoccurs.

Accordingly, the desire to detect enzyme analytes and to use enzymelabels as a diagnostic tool in various analytical test systems has givenrise to the development of optical indicator compounds for use in thedetection and measurement of the activity of such enzymes. Typically,such known optical indicator compounds comprise a detectable chemicalgroup, such as a fluorogen or a chromogen, which has been derivatizedwith an enzyme cleavable substrate group specific for the enzyme ofinterest. Such optical indicator compounds exhibit an optical signalwhich is different from the optical signal which is provided by thecleaved native form of the fluorogen or chromogen. In principle, theenzyme cleaves the indicator compound to liberate the fluorogen orchromogen in the form of a distinctly fluorescent or colored product toprovide a change in fluorescence or color which is proportional to theamount of enzyme present which, in turn, can be correlated to the amountof analyte present in a liquid test sample.

In particular, the detection and/or determination of hydrolases, i.e.,enzymes which catalyse hydrolysis reactions of esters, glycosidic bonds,peptide bonds, other carbon-nitrogen bonds, and acid anhydrides [seeLehninger, Biochemistry (Worth Publishers, Inc, New York, N.Y., 1970) p.148], is of interest in the diagnosis and monitoring of various diseasessuch as, for example, the determination of amylase and lipase in thediagnosis of pancreatic disfunction [see Kaplan and Pesce, ClinicalChemistry--Theory, Analysis and Correlation (C. V. Mosby Co., St. Louis,Mo., 1984) Chapter 56], determination of N-acetylglucosaminidase (NAG)as an indicator of renal disease [see Price, Curr. Probl. Clin. Biochem.9, 150 (1979)] and detection of esterase as an indicator for leukocytes[see Skjold, Clin. Chem. 31, 993 (1985)].

Enzymes have also gained importance in the diagnostic as well as thebiotechnology fields. For example, alkaline phosphatase andβ-D-galactosidase have found increasing use as indicator enzymes forenzyme immunoassays [see Annals of Clinical Biochemistry 16, 221-40(1979)]. Accordingly, the use of enzymes such as glycosidases,particularly β-D-galactosidase, as indicator enzyme labels in analyticaltest systems has given rise to the development of substrate glycosidessuch as phenyl-β-D-galactoside, o-nitrophenyl-β-D-galactoside andp-nitrophenyl-β-D-galactoside [see Biochem. Z., Vol. 33, p. 209 (1960)]which are hydrolysed by β-D-galactosidase to liberate the phenols whichare determined photometrically in the ultraviolet range, or thenitrophenols which are determined in the shortwave visible range,respectively. A few other examples are the chromogenic resorufinderivatives of European Patent application No. 156,347, and thechromogenic acridinone derivatives of European Patent Application No.270,946.

The use of β-D-galactosides has also been described in conjunction withhistochemical investigations, such as the naphthyl-β-D-galactosidesdescribed in Histochemie, Vol. 35, p. 199 and Vol. 37, p. 89 (1973), andthe 6-bromo-α-naphthyl derivatives thereof described in J. Biol. Chem.,Vol. 195, p. 239 (1952). According to such test systems, the naphtholswhich are liberated upon the interaction of the galactoside with theenzyme are reacted with various diazonium salts to yield the respectiveazo-dyes which can then be visualized.

There continues to be a need for new compounds having desirablecombinations of chromogenic substrate properties such as extinctioncoefficient, absorbance maxima, water solubility, color shift, andturnover rate.

Merocyanine dyes have previously been used as analytical reagents,although not as chromogenic enzyme substrates. European PatentApplication No. 47470 describes the use of cyanine and merocyanine dyesas labels for antibodies or antigens in an immunochemical assay. Thelabeled antigen or antibody is subjected to an immune reaction andcontacted with a silver halide which is then exposed to light anddeveloped. The resulting optical density is measured. PCT PublicationNo. 86-06374 describes a conjugate of a highly fluorescent merocyaninedye with a biologically active moiety useful in diagnostic assays. Theapplication uses dye-labeled antibodies to measure analytes in a testsample. Kiciak [Roczniki Chemii 37,225(1963)] describes the preparationof a merocyanine acetate ester but gives no suggestion that it mightfunction as a chromogenic enzyme substrate.

SUMMARY OF THE INVENTION

The present invention provides novel chromogenic merocyanine enzymesubstrate compounds of the general formula (A): ##STR2## where Yrepresents an enzymatically cleavable group that is selected to conferspecificity for the corresponding enzyme of analytical interest.Further, A represents a nonmetallic atomic group or residue whichcompletes a 5- or 6-membered carbocyclic or heterocyclic ring or a fusedring system consisting of 5- and/or 6-membered heterocyclic orcarbocyclic rings; B represents a nonmetallic atomic group or residuewhich completes a 5- or 6-membered N-containing heterocyclic ring or afused ring system consisting of 5- and/or 6-membered heterocyclic orcarbocyclic rings; R¹ is alkyl or aryl; R² and R³, which may be the sameor different, are hydrogen or lower alkyl; m, n, and p, which may be thesame or different, are integers from 0 through 3 provided that m+n+p isat least 2; and X is a counterion (anion). The ring system formed withthe group A in the formula is referred to herein as the acidic nucleus,and that formed with the group B as the basic nucleus.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 and 1a are tables of some representative basic and acidic nucleithat can form merocyanines.

FIGS. 2 through 5 are flow diagrams of the principal steps in thepreferred convergent synthesis of substrate compounds as described inthe Examples.

FIGS. 6 through 8 are graphs of the dose response of some substratecompounds of the present invention to the enzyme β-galactosidase.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The merocyanines are a class of sensitizing dyes discoveredindependently in the early 1930's by Kendall (British Pat. Nos. 426,718;428,222; 428,359; 428,360; 432,628; 549,201-4; 555,549; 555,550;624,027; 624,951; and 634,952) and Brooker (U.S. Pat. Nos. 2,078,233;2,089,729; 2,153,169; 2,161,331; 2,165,219; 2,165,338; 2,170,803-7;2,177,401-3; 2,185,182; 2,185,343; 2,186,624; 2,211,762; and 2,332,433).The literature on these compounds has been the subject of severalreviews--Quarterly Reviews 4,327(1950); "The Chemistry of SyntheticDyes", vol. II, by K. Venkataraman, Academic Press, New York (1952),Chapter 38; "The Cyanine Dyes and Related Compounds" by F. Hamer,Interscience Publishers, New York (1964), chapters 10, 11 and 14; "TheChemistry of Synthetic Dyes, vol. IV, ed. K. Venkataraman, AcademicPress, New York (1971), Chapter 5; and "The Chemistry of HeterocyclicCompounds", vol. 30, ed. E. Taylor and A. Weissberger,Wiley-Interscience, New York (1977).

Merocyanines are composed of an acidic nucleus and a basic nucleus asrepresented by the general formula (B) ##STR3## The chromophore in thismolecule is the dipolar amidic system represented in formula (C).##STR4## A simple merocyanine is defined as one in which the nuclei aredirectly linked, i.e., m=0 in formula (B) and a dimethinmerocyanine asone in which m=1 ("The Cyanine Dyes and Related Compounds", supra).Dimethinmerocyanines have also been called merocarbocyanines("Kirk-Othmer Encyclopedia of Chemical Technology", vol. 7, 3rd ed.,Wiley-Interscience, New York (1979), pp. 335-358). Homologs in which m=2and 3 are also known. Synthetic methods for the preparation of thisclass of compounds have been recently reviewed ("The Chemistry ofHeterocyclic Compounds", supra).

Among the merocyanine dyes, those of the stilbazolium betaine type[formula (D)] have found continuing interest because of theirsolvatochromatic properties. ##STR5## As early as 1920, a compound ofthis type was reported to function as a pH indicator, being"lemon-yellow" in the presence of acids and being "blood red" in thepresence of alkali [L. F. Werner, J. Am. Chem. Soc. 42,2309(1920)]. Overthe years, other pH indicators were recognized within this class of dyes[Helv. 23,247(1940); Ukran. Khim. Zhur. 18,347(1952); Farmacia(Bucharest) 22,345(1974)]. When the oxygen functionality of the acidicnucleus is derivatized, a shift in color from the underivatized dye hasbeen noted [J. Org. Chem. 14,302(1949); Roczniki Chemii 37,225(1963)].

It is an object of this invention to prepare merocyanine dyes in whichthe oxygen functionality of the acidic nucleus has been derivatized withan enzyme cleavable group and to determine whether such compounds haveutility as chromogenic enzyme substrates. As a result, it has been foundthat compounds of formula (A) are advantageous chromogenic enzymesubstrates.

As used herein "alkyl" is intended to include linear and branched formsof unsubstituted hydrocarbon residues of the general formula--C_(n)H_(2n+1), preferably of the "lower alkyl" aliphatic type wherein n is 6or less, such as methyl, ethyl, n-propyl, iso-propyl, n-butyl,iso-butyl, tert-butyl, n-hexyl, and the like, as well as substitutedforms thereof.

Further, "aryl" is intended to include organic residues derived from anaromatic hydrocarbon ring or ring system by removal of a hydrogen atom,and include the unsubstituted hydrocarbon ring residues such as phenyland naphthol, and substituted forms thereof. For purposes of the presentinvention, aryl residues include those bearing one or more same ordifferent functional groups or substituents which can be selected by oneskilled in the art to provide the chromogenic enzyme substrate compoundsof the present invention.

More particularly, where "aryl" and "alkyl" are substituted, suchsubstitution is intended to include such groups or substituents whenmono- or polysubstituted with functional groups which do notsubstantially negate the useful features of the present compounds. Suchfunctional groups include chemical groups which may be introducedsynthetically and result in the stable and useful chromogenic enzymesubstrate indicator compounds of the present invention. Examples of suchfunctional groups include, but are not intended to be limited to, halo(e.g., fluoro, chloro, bromo), substituted amino such as dialkylamino,nitro, alkoxy, aryloxy, alkyl, aryl, cyano, sulfo, carboxy, andalkoxycarbonyl.

ENZYMATICALLY-CLEAVABLE GROUPS

According to the present invention, the enzymatically-cleavable group Yis a radical of a compound Y-OH comprising an enzyme-specific moiety toprovide novel chromogenic enzyme substrate compounds which conferspecificity to a wide variety of enzymes encountered in a clinicalchemistry, particularly hydrolases. The compound Y-OH is intended toinclude, but is not necessarily limited to, sugars and derivativesthereof, acyl groups including aliphatic and aromatic carboxylic acids,amino acids and peptides, and inorganic acids such as phosphoric andsulfuric acid groups.

It is to be understood that it will be evident to one skilled in the artthat the selection of the enzymatically-cleavable group Y will depend,of course, upon the particular enzyme of interest. For example, wherethe enzyme of interest is a glycosidase, a glycoside can be prepared inwhich the enzymatically-cleavable group Y is the glycosidic radicalcorresponding to the natural substrate for the particular glycosidase.Suitable glycosidic radicals include, but are not intended to be limitedto, mono- and oligosaccharide radicals, which are capable of beingincorporated into a glycoside substrate specific for a particularglycosidase enzyme and cleaved by said enzyme, such as radicals ofβ-D-galactopyranose, α-D-galactopyranose, β-D-glucopyranose,α-D-glucopyranose and α-D-mannopyranose, as well as amino sugars such asN-acetylglucosamine and N-acetylneuraminic acid, and the like radicals.Other suitable glycosidic radicals include oligosaccharide chains frombetween about 2 to 20, preferably 2 to 7, monosaccharide units attachedby α-1→4 glucosidic linkages, which can be broken down bysaccharide-chain splitting enzymes to a mono- or oligosaccharide which,in turn, can be cleaved by a corresponding glycosidase, such as, forexample, radicals of maltopentose, maltohexose and maltoheptose.

It is to be understood that in some instances where the glycosidicradical is an oligosaccharide chain as heretofore described, such chainis first modified or broken down to a shorter oligosaccharide ormonosaccharide by the enzyme under determination to produce a secondarysubstrate compound in which the enzymatically-cleavable group is cleavedfrom the merocyanine indicator group by a secondary enzyme, in whichcase the secondary compound is then contacted with the secondary enzymeto generate a measurable change in absorbance as heretofore described.For example, where the enzyme under determination is α-amylase, theoligosaccharide chain is cleaved to produce a secondary glycosidesubstrate compound, e.g., an α-glucoside or β-glucoside, in which theresulting glycoside group thereof is cleavable from the merocyanineindicator group by a secondary glycosidase enzyme, e.g., α-glucosidaseor β-glucosidase, respectively.

In the case of nonspecific esterase enzymes, the enzymatically-cleavablegroup Y is an acyl radical group of the formula ##STR6## Where V islower alkyl or aryl, such compounds can be employed for the detection ofnonspecific esterase enzymes such as cholinesterase, acylase, lipase,and the like.

The chromogenic enzyme substrate compounds of the present invention canalso be utilized for the detection of proteolytic enzymes commonly foundin leukocytes. Such compounds are esters of the general formula where Yis a radical of the compound Y-OH and where Y-OH is an N-protected aminoacid or short peptide, e.g., consisting of between about 2 to 5 aminoacid units. For example, Y can be an N-protected amino acidN-tosyl-L-alanine radical. It will be appreciated that the presentinvention contemplates other carboxylic acid residues, amino acidresidues and N-protecting groups as equivalents, as will be described ingreater detail hereafter.

Similarly, for the detection of alkaline phosphatase from a liquid testsample, the enzymatically-cleavable group Y is a radical of the compoundY-OH wherein Y-OH is a phosphoric acid group.

BASIC AND ACIDIC NUCLEI

An extremely wide variety of different substituted and unsubstitutedbasic and acidic nuclei can be used to form the merocyanine dyecomponent of the present compounds. Merocyanines reported in theliterature are exemplified by those combinations of the basic and acidicnuclei depicted in FIG. 1 that are listed in Table A. These can be usedin forming the present compounds where --OY will be substituted for --OHon the acidic nucleus. Other examples of merocyanine dyes are found inthe various reviews, patents, and other literature cited herein.

BASIC NUCLEI

One skilled in the art of dye synthesis will recognize that virtuallyany basic nucleus known can be incorporated into the present compounds.The basic nucleus will fundamentally be a 5- or 6-membered N-containingheterocyclic ring or a fused ring system consisting of 5- and/or6-membered heterocyclic or carboxyclic rings. Accordingly, in formula(A), B represents an appropriate residue to complete such basic nuclei.Representative of suitable nonmetallic atomic groups are C, S, O, N, andSe. The 5- or 6-membered heterocyclic rings are rings consisting ofcarbon atoms and one or more heteroatoms selected from N, O, S or Sejoined by single and/or double bonds, and are represented by the nucleishown in Table B.

                  TABLE A                                                         ______________________________________                                        Merocyanine Dye                                                               (Basic Nucleus-                                                               Acid Nucleus) Literature Reference                                            ______________________________________                                        1-A           J. Chem. Soc., 3313 (1955); J. Org.                                           Chem. 14, 302 (1949); J. Am. Chem.                                            Soc. 73, 5350 (1951); J. Gen. Chem.                                           USSR 17, 1468 (1947); Farmacia                                                (Bucharest) 22, 345 (1974); J.                                                Chem. Ed. 54, 709 (1977).                                       1-D           Farmacia (Bucharest) 22, 345 (1974).                            1-I           Ann. 592, 161 (1955).                                           1-K           Ann. 592, 161 (1955).                                           1-M           J. Am. Chem. Soc. 73, 5350 (1951).                              2-A           J. Chem. Soc., 3313 (1955).                                     2-C           J. Chem. Soc., 3038 (1951).                                     2-D           Farmacia (Bucharest) 22, 345 (1974).                            2-F           J. Chem. Soc., 3038 (1951).                                     2-I           Ukran. Khim. Zhur. 18, 347 (1952).                              2-O           J. Am. Chem. Soc. 73, 5356 (1951).                              3-J           Helv. 23, 247 (1940).                                           4-A           J. Chem. Soc., 3313 (1955); Chem.                                             Ber. 74, 471 (1941); Ann. 592, 161                                            (1955); Farmacia (Bucharest) 22,                                              345 (1974).                                                     4-I           J. Gen. Chem. USSR 17, 1468 (1947).                             4-K           J. Gen. Chem. USSR 17, 1468 (1947).                             5-A           J. Am. Chem. Soc. 42, 2309 (1920);                                            J. Gen. Chem. (USSR) 10, 600 (1940);                                          Chem. Ber. 74, 471 (1941); Roczniki                                           Chemii 37, 225 (1963).                                          5-B           J. Am. Chem. Soc. 42, 2309 (1920).                              5-C           J. Chem. Soc., 3038 (1951).                                     5-D           J. Am. Chem. Soc. 42, 2309 (1920);                                            J. Gen. Chem. (USSR) 10, 600 (1940).                            5-F           J. Chem. Soc., 3038 (1951).                                     5-G           Ukran. Khim. Zhur. 18, 347 (1952).                              5-I           Ukran. Khim. Zhur. 18, 347 (1952).                              5-J           Helv. 23, 247 (1940).                                           5-L           J. Chem. Soc., 2135 (1952).                                     5-N           J. Am. Chem. Soc. 73, 5332 (1951).                              6-D           Helv. 23, 247 (1940).                                           6-E           Helv. 23, 247 (1940).                                           6-I           Ann. 592, 161 (1955).                                           6-J           Helv. 23, 247 (1940).                                           7-A           J. Am. Chem. Soc. 73, 5330 (1951).                              7-C           J. Chem. Soc., 3038 (1952).                                     7-D           Helv. 23, 247 (1940).                                           7-E           Helv. 23, 247 (1940).                                           7-I           Ukran. Khim. Zhur. 18, 347 (1952).                              7-J           Helv. 23, 247 (1940).                                           7-L           J. Chem. Soc. 2135 (1952).                                      8-A           J. Am. Chem. Soc. 73, 5350 (1951).                              8-N           J. Am. Chem. Soc. 73, 5332 (1951).                              9-D           Helv. 23, 247 (1940).                                           9-E           Helv. 23, 247 (1940).                                           9-J           Helv. 23, 247 (1940).                                           10-J          J. Chem. Soc., 3038 (1951).                                     11-A          J. Gen. Chem. (USSR) 10, 600 (1940);                                          Farmacia (Bucharest) 22, 347 (1974).                            11-D          J. Gen. Chem. (USSR) 10, 600 (1940).                            11-I          Ukran. Khim. Zhur. 18, 347 (1952).                              12-A          J. Gen. Chem. (USSR) 10, 600 (1940);                                          Farmacia (Bucharest) 22, 347 (1974).                            12-I          Ukran. Khim. Zhur. 18, 347 (1952).                              13-I          Ukran. Khim. Zhur. 18, 347 (1952).                              13-L          J. Chem. Soc. 2135 (1952).                                      14-A          J. Am. Chem. Soc. 73, 5350 (1951).                              15-A          J. Gen. Chem. (USSR) 10, 600 (1940);                                          J. Am. Chem. Soc. 73, 5350 (1951);                                            Ukran. Khim. Zhur. 18, 347 (1952);                                            Roczniki Chemii 37, 225 (1963);                                               Farmacia (Bucharest) 22, 345 (1974).                            15-C          J. Chem. Soc., 3038 (1951).                                     15-D          J. Gen. Chem. (USSR) 10, 600 (1940).                            15-I          Ukran. Khim. Zhur. 18 347 (1952).                               15-J          Helv. 23, 247 (1940).                                           15-L          J. Chem. Soc. 2135 (1952).                                      15-N          J. Am. Chem. Soc. 73, 5332 (1951).                              16-A          J. Am. Chem. Soc. 73, 5350 (1951).                              17-A          J. Am. Chem. Soc. 73, 5350 (1951).                              18-H          J. Gen. Chem. USSR 17, 1468 (1947).                             18-L          J. Gen. Chem. USSR 17, 1468 (1947).                             19-A          Ukran. Khim. Zhur. 18, 347 (1952).                              19-I          Ukran. Khim. Zhur. 18, 347 (1952).                              19-L          J. Chem. Soc., 2135 (1952).                                     20-I          Ukran. Khim. Zhur. 18, 347 (1952).                              20-L          J. Chem. Soc., 2135 (1952).                                     21-J          Helv. 23, 247 (1940).                                           21-L          J. Chem. Soc., 2135 (1952).                                     ______________________________________                                           The nuclei shown in Table B are merely a few examples of useful rings     and ring systems for the basic nuclei. It will be evident that others can     also be used for this purpose as well as various derivative and     substituted forms of the nuclei depicted.

A particularly preferred basic nucleus is of formula (E) ##STR7##wherein Z is disubstituted methylene, e.g., di(lower alkyl)methylene,vinylene, alkyl- or aryl-substituted N, e.g., lower alkyl or phenylsubstituted N, O, S, or Se, R¹ is as described herein, and wherein thephenyl group depicted in the formula is substituted or unsubstituted.The basic nucleus can be unsubstituted or bear substituents of such typeand of such number on a given nucleus which will not interfersubstantially with the ultimate chromogenic substrate propertiesdesired. Such substituents will be recognized to include alkyl,particularly lower alkyl, aryl, particularly phenyl and substitutedphenyl, alkoxy, aryloxy, halo, nitro and amino or substituted amino,e.g., dialkylamino, cyano, sulfo, carboxyl, carboxyalkyl, carboxamide,and carboxamidoalkyl.

                  TABLE B                                                         ______________________________________                                        (1) 5-membered heterocyclic rings                                              ##STR8##        Z = O, S, Se, NR.sup.1 W.sup.1, W.sup.2 = H, alkyl, aryl                      .sup.1 = alkyl                                                ##STR9##                                                                     (2) 6-membered heterocyclic rings                                              ##STR10##       W.sup.3 = H, alkyl, aryl R.sup.1 = alkyl                      ##STR11##                                                                    (3) fused heterocyclic-2 ring system                                           ##STR12##       Z = NR.sup.1, O, S, Se, CH.sub.2, C(CH.sub.3).sub.2                           W.sup.4 = H, alkyl, aryl, alkoxy, NR.sub.2.sup.1,                             NO.sub.2, halo, cyano, aryloxy R.sup.1 = alkyl, aryl,                         carboxyalkyl, sulfoalkyl                                      ##STR13##                                                                     ##STR14##                                                                    (4) fused heterocyclic-3 ring system                                           ##STR15##       Z = S, O, Se, NR.sup.1 R.sup.1 = alkyl, aryl,                                 carboxyalkyl, sulfoalkyl                                      ##STR16##                                                                     ##STR17##       Z = aminoalkyl R.sup.1 = alkyl, aryl, carboxy- alkyl,                         sulfoalkyl                                                    ##STR18##                                                                     ##STR19##                                                                    ______________________________________                                    

Representative examples of basic nuclei are thiazole, 4-methylthiazole,4-phenylthiazole, 4,5-dimethylthiazole, 4,5-diphenylthiazole,benzothiazole, 4-chlorobenzothiazole, 5-chlorobenzothiazole,6-chlorobenzothiazole, 7-chlorobenzothiazole, 5-nitrobenzothiazole,6-nitrobenzothiazole, 4-methylbenzothiazole, 5-methylbenzothiazole,6-methylbenzothiazole, 5-bromobenzothiazole, 6-bromobenzothiazole,5-iodobenzothiazole, 5-phenylbenzothiazole, 5-methoxybenzothiazole,6-methoxybenzothiazole, 5-ethoxybenzothiazole,5-ethoxycarbonylbenzothiazole, 5-phenethylbenzothiazole,5-fluorobenzothiazole, 5-chloro-6-nitrobenzothiazole,5-trifluoromethylbenzothiazole, 5,6-dimethylbenzothiazole,tetrahydrobenzothiazole, 4-phenylbenzothiazole, 5-phenylbenzothiazole,naphtho(2,1-d)thiazole, naphtho(1,2-d)thiazole, naphtho(3,4-d)thiazole,5-methoxynaphtho(1,2-d)thiazole, 7-ethoxynaphtho(2,1-d)thiazole,8-methoxynaphtho(2,1-d)thiazole, 5-methoxynaphtho(3,4-d)thiazole,oxazole, 4-methyloxazole, 4-nitrooxazole, 5-methyloxazole,4-phenyloxazole, 4,5-diphenyloxazole, 4-ethyloxazole, benzoxazole,5-chlorobenzoxazole, 5-methylbenzoxazole, 5-bromobenzoxazole,5-fluorobenzoxazole, 5-phenylbenzoxazole, 5-methoxybenzoxazole,5-trifluoromethylbenzoxazole, 5-nitrobenzoxazole, 5-methylbenzoxazole,6-chlorobenzoxazole, 6-nitrobenzoxazole, 6-methoxybenzoxazole,6-hydroxybenzoxazole, 5,6-dimethylbenzoxazole, 4,6-dimethylbenzoxazole,5-ethoxybenzoxazole, naphtho(2,1-d)oxazole, naphtho(1,2-d)oxazole,naphtho(3,4-d)oxazole, 5-nitronaphtho(3,2-d)oxazole, 4-methylselenazole,4-nitroselenazole, 4-phenylselenazole, benzoselenazole,5-chlorobenzoselenazole, 5-nitrobenzoselenazole,5-methoxybenzoselenazole, 5-hydroxybenzoselenazole,6-nitrobenzoselenazole, 5-chloro-6-nitrobenzoselenazole,naphtho(2,1-d)selenazole, naphtho(1,2-d)selenazole,3,3-dialkylindolenine and ring substituted 3,3-dialkylindolenines,1-alkylimidazole, 1-alkyl-4-phenylimidazole, 1-alkylbenzimidazole,1-alkyl-5-methoxybenzimidazole, 1-alkyl-5-cyanobenzimidazole,1-alkyl-5-fluorobenzimidazole, 1-alkyl-5-trifluoromethylbenzimidazole,1-alkylnaphtho(1,2-d)imidazole, 1-aryl-5,6-dichlorobenzimidazole,1-aryl-5-chlorobenzimidazole, 1-arylimidazole, 1-arylbenzimidazole,1-aryl-5-chlorobenzimidazole, 1-aryl-5,6-dichlorobenzimidazole,1-aryl-5-methoxybenzimidazole, 1-aryl-5-cyanobenzimidazole,1-arylnaphtho(1,2-d)imidazole, wherein the alkyl group is methyl, ethyl,propyl, isopropyl, butyl, 2-hydroxyalkyl, 3-hydroxypropyl, and the like,and the aryl group is phenyl, a substituted phenyl, a methyl-substitutedphenyl or a methoxy-substituted phenyl. Examples of the basic nucleusfurther include 2-pyridine, 4-pyridine, 5-methyl-2-pyridine,3-methyl-4-pyridine, and quinoline nuclei, (e.g., 2-quinoline,3-methyl-2-quinoline, 5-ethyl-2-quinoline, 6 -methyl-2-quinoline,6-methoxy-2-quinoline, 8-chloro-2-quinoline, 4-quinoline,6-ethoxy-4-quinoline, 6-nitro-4-quinoline, 8-chloro-4-quinoline,8-fluoro-4-quinoline, 8-methyl-4-quinoline, and 8-methoxy-4-quinoline).

Particularly preferred basic nuclei are the substituted andunsubstituted forms of indolenine, naphthothiazole, benzoxazole,benzothiazole, quinoline, thiazole, rhodanine, benzoselenazole, andbenzimidazole, and particularly include 4-methylthiazole,4-phenylthiazole, benzothiazole, 5-chlorobenzothiazole,5-methylbenzothiazole, 6-methoxybenzothiazole, naphtho(2,1-d)thiazole,naphtho(1,2-d)thiazole, benzoxazole, 5-methylbenzoxazole,benzoselenazole, 3,3-dimethylindolenine, 4-quinoline, 2-quinoline,6-methoxy-2-quinoline and 4-pyridine.

Substituent R¹ on the basic nucleic as depicted in formula (A) cangenerally be alkyl or aryl as defined above, and preferably issubstituted or unsubstituted lower alkyl or phenyl. Examples, withoutlimitation, are methyl, ethyl, propyl, butyl, benzyl, phenyl,β-phenyethyl, 1-hydroxyethyl, 2-methoxyethyl, 2-(2-methoxyethoxy)-ethyl,carboxymethyl, 2-carboxyethyl, 3-carboxypropyl, 4-carboxybutyl,2-sulfoethyl, 3-sulfopropyl, 3-sulfobutyl, 4-sulfobutyl,2-(pyrrolidin-2-on-1-yl)ethyl, tetrahydrofurfuryl, 2-acetoxyethyl,carbomethoxymethyl, and 2-methanesulfonylaminoethyl.

As depicted in formula (A), and implied when describing cationic basicnuclei as in FIG. 1, the merocyanine compounds have a correspondingcounterion (anion) X. The nature of the counterion, whether the compoundof formula (A) is in an ionized or nonionized form, is believed not tobe critical to the present invention, although solubility may beaffected by the nature of the counter ion (see The Chemistry ofSynthetic Dyes, vol. 4, supra, p. 294). Accordingly, such counter ioncan take a wide variety of forms. Just a few of the commonly foundcounter ions (which complex with the merocyanine from the reactionmixture in which they are synthesized or the solutions in which they aredissolved) are chloride, bromide, iodide, tetrafluoroborate,trifluoroacetate, acetate, sulfate, tosylate, phosphate, andperchlorate.

ACIDIC NUCLEI

As in the case of the basic nucleus, the acidic nucleus can vary widelyas is known in the art (FIG. 1, Table A, and cited references, supra).The acidic nucleus will fundamentally be a 5- or 6-membered carbocyclicor heterocyclic ring or a fused ring system consisting of 5- and/or6-membered carbocyclic or heterocyclic rings. Accordingly, in formula(A), A represents an appropriate residue to complete such acidic nuclei.Representative of suitable nonmetallic atomic groups are C, S, O, N andSe. As 5- or 6-membered carbocyclic rings are intended rings consistingof 5 or 6 carbon atoms joined by single and/or double bonds. As 5- or6-membered heterocyclic rings are intended rings consisting of carbonatoms and one or more heteroatoms selected from N, O, S or Se joined bysingle and/or double bonds. Table C depicts some representativecarbocyclic and heterocyclic rings and fused ring systems that can serveas the acidic nuclei. The depicted structures are merely a few examplesof particular acidic nuclei. It will be evident that others can also beused for this purpose as well as various derivative and substitutedforms of the nuclei depicted.

Particularly preferred acidic nuclei are substituted or unsubstituted1,2-naphthylene, 1,4-phenylene, 1,4-naphthylene, and 2,6-naphthylene.

Representative examples of acidic nucleic are 3-alkyl-rhodanine,3-arylrhodanine, 1-alkyl-2-pyrazolin-5-one, 3-aryl-5-oxazolone,1,3-dialkyl-2-thiohydantoin (1,3-dialkyl-2-thio-2,4-imidazolidinedine),1,3-diaryl-2-thiohydantoin (1,3-diaryl-2-thio-2,4-imidazolidinedionenucleus), 1,3-dialkyl-2-thiobarbituric acid, 3-alkyl-4-thiazolidinone,3-aryl-4-thiazolidinone, indan-1,3-dione, thioindoxyl,1,3-dialkylhydantoin (1,3-dialkyl-2,4-imidazolidinedione nucleus),1,3-diarylhydantoin (1,3-diaryl-2,4-imidazolidinedione nucleus),4-hydroxyphenyl, 4-hydroxy-3-methoxyphenyl, 4-hydroxy-3-nitrophenyl,2-hydroxyphenyl, 2-hydroxy-3-methoxyphenyl, 2-hydroxy-3-nitrophenyl,2-hydroxy-4-nitrophenyl, 2-hydroxy-5-ethoxyphenyl,2-hydroxy-5-dimethylaminophenyl, 4-hydroxy-1-naphthyl,2-hydroxy-1-naphthyl, 9-hydroxy-10-anthryl, 4-hydroxy-1-anthryl,6-hydroxy-2-naphthyl and 5-hydroxy-1-naphthyl. Preferred acidic nucleiinclude 4-hydroxy-1-phenyl, 4-hydroxy-3-nitrophenyl, 2-hydroxyphenyl,4-hydroxy-1-naphthyl, 2-hydroxy-1-naphthyl, 6-hydroxy-2-naphthyl,5-hydroxy-1-naphthyl and 4-hydroxy-1-anthryl.

                  TABLE C                                                         ______________________________________                                        (1) 4-membered rings                                                           ##STR20##         W.sup.5 = alkyl                                            (2) 5-membered rings                                                           ##STR21##         R.sup.1 = alkyl, aryl, carboxyalkyl, sulfoalkyl             ##STR22##                                                                     ##STR23##                                                                     ##STR24##                                                                     ##STR25##                                                                    (3) 6-membered rings                                                           ##STR26##         W.sup.6 = alkyl, aryl, alkoxy, aryloxy, cyano, halo,                          nitro, carboxycarbonyl                                      ##STR27##                                                                     ##STR28##                                                                    (4) fused ring-2 ring system                                                   ##STR29##         Z = S, carbonyl W.sup.6 = alkyl, aryl, alkoxy,                                aryloxy, cyano, halo, nitro, carboxycarbonyl                ##STR30##                                                                     ##STR31##                                                                     ##STR32##                                                                    (5) fused ring-3 ring system                                                   ##STR33##         W.sup.6 = alkyl, aryl, alkoxy, aryloxy, cyano, halo,                          nitro, carboxycarbonyl                                     ______________________________________                                    

The basic and acidic nuclei are joined, as depicted in formula (A) by asingle bond (m=0) or conjugated vinylene groups (m=1-3).Dimethinmerocyanines, where m=1, are most common and preferred. Geometryabout this double bond is usually trans, but the cis orientation is alsocontemplated. Where m=1, the bridge carbons can be substituted orunsubstituted, e.g., R² and R³ can be the same or different and can behydrogen, lower alkyl, or cyano.

A particularly preferred class of the merocyanine enzyme substratecompounds of the present invention are represented by the formula (F)##STR34## wherein Y is an enzymatically-cleavable group which is aradical of a compound Y-OH selected from sugars and derivatives thereof,aliphatic and aromatic carboxylic acids, amino acids, peptides,phosphoric acid, and sulfuric acid; B represents a non-metallic atomicgroup or residue which completes a 5- or 6-membered N-containingheterocyclic ring or a fused ring system consisting of three or less 5-and/or 6-membered heterocyclic or carbocyclic rings; R¹ is substitutedor unsubstituted lower alkyl or aryl, e.g., phenyl; Ar is substituted orunsubstituted phenylene, naphthylene or anthrylene; n is an integer from1 through 3; and X is a counterion (anion). Most commonly Y-OH will beα-D-galactose, β-D-galactose, α-glucose, β-glucose, α-mannose,N-acetylglucosamine, N-acetylneuraminic acid, or an oligosaccharidechain of from between about 2 to 20 monosaccharide units e.g.,maltopentose, maltohexose, and maltoheptose. In the compounds of formula(F), Ar is preferably substituted or, more usually, unsubstituted1,2-naphthylene, 1,4-phenylene, 1,4-naphthylene, or 2,6-naphthylene, andB completes a residue of substituted or unsubstituted indolenium,β-naphthothiazolium, benzoxazolium, benzothiazolium, quinolium,thiazolium, or rhodaninium, and more preferably is of formula (E).

Compounds that are particularly useful are of formula (G) ##STR35##wherein Y is a radical of a compound Y-OH selected from sugars andderivatives thereof, particularly β-D-galactose; Ar is 1,4-phenylene,1,4-naphthylene or 2,6-naphthylene; R¹ is substituted or unsubstitutedlower alkyl, particularly ethyl or methyl; Z is di(loweralkyl)methylene, vinylene, O, S, or Se, and wherein the phenyl ring issubstituted or unsubstituted and X is a counterion (anion).

SYNTHESIS

Dye synthesis in general has been characterized in the literature asbeing of the condensation type, that is two intermediates reacting undersuitable conditions with elimination of some simple molecule. Thisgeneral description of the combining of nucleophilic and electrophilicreagents covers most methods of non-oxidative dye synthesis. Typically,the nucleophile is a methylene base derived from an active methylquaternary salt, and the electrophile is an orthoester or aldehyde.Coupling of an active methyl quaternary salt (basic nucleus) with anaromatic aldehyde (acidic nucleus) under basic conditions is a commonmethod used in the preparation of merocyanine dyes. Other methods areknown as well and described in the literature cited herein.

In principle, one can first prepare a merocyanine dye with an availablehydroxyl group on the acidic nucleus for subsequent modification to formthe enzymatically cleavable group. In practice, however, this has beenfound to be generally unsuccessful, the condensed merocyanine dye beingsubstantially unreactive to form the enzymatically cleavable group. Thisis likely due to the merocyanine being in the uncharged or neutraltautomeric form [see formula (B)] under the basic conditions requiredfor condensation of the enzymatically cleavable group precursor.

Accordingly, a convergent synthesis has been devised in which the basicnucleus is condensed with an acidic nucleus that has already beenmodified to comprise the enzymatically cleavable group. As the firststep in the synthesis, a class of aryl aldehyde intermediates are formedby reaction of a hydroxyl-functionalized arylaldehyde under appropriateconditions to incorporate the appropriate enzynmatically cleavablegroup. The resulting compounds will be of formula (H) ##STR36## whereinA, Y and p are described herein above and R⁴ is hydrogen or lower alkyl.Particularly novel are the intermediates of formula (J)

    OHC--Ar--O--Y                                              (J)

wherein Ar is substituted or unsubstituted phenylene, naphthylene,particularly 1,4-phenylene, 1,4-naphthylene or 2,6-naphthylene.

To prepare the present substrate compounds, such arylaldehydes arereacted with a quaternary salt derivative of formula (K) ##STR37##wherein B, R¹, n and X are as described above and R⁵ is hydrogen, loweralkyl, or cyano, under appropriate basic conditions as are known in theart. Generally, arylaldehydes of the formula (J) are first dissolved orsuspended in a suitable basic solvent, basic solvent mixture or solventcontaining a base, which is capable of at least partially dissolving thearylaldehyde. Such basic solvents include pyridine, quinoline,piperidine, pyrrolidine, hexamethylphosporamide and di- andtrialkylamines. Mixtures of these basic solvents with other solvents,including alcohols such as methanol and ethanol, ethers such astetrahydroguran and dioxane, amides such as dimethylformamide anddimethylacetamide, aromatics such as toluene and benzene, haloalkanessuch as chloroform and dichloromethane, ketones such as acetone andmethylethylketone, and esters such as ethyl acetate, are also useful.Additionally, certain alkoxide bases such as sodium methoxide, sodiumethoxide and potassium tert-butoxide will be useful in alcoholicsolvents such as methanol, ethanol and tert-butanol. A preferred solventis pyridine. The solution or suspension of the arylaldehyde of theformula (J) is then treated, either at once or in portions over a periodof from 10 minutes to 5 hours, preferably 0.25 to 2.25 hours, with 0.5to 5.0 molar equivalents, preferably 1.0 to 1.5 molar equivalents, ofquaternary salt of the formula (K). The reaction mixture is maintainedat a temperature of 0° C. to 150° C., preferably 50° C. to 100° C., fora period of time from 1 minute to 36 hours, preferably 5 to 20 hours,then the solvent is removed under reduced pressure and the compound ofthe formula (E) is purified using methods known in the art, such aschromatography.

Preparation of the enzymatically cleavable group-modified arylaldehydeswill proceed as appropriate for the cleavable group involved.

Glycosides of the reactive acidic nucleus can be prepared according tomethods known in the art of carbohydrate chemistry employing knownderivatives of carbohydrates of the formula Y-OH which are reacted withan appropriate acidic nucleus. Such carbohydrate derivatives, which insome instances carry protecting groups, are commercially available(Aldrich Chemical Co., Milwaukee, Wis., U.S.A.; Sigma Chemical Co., St.Louis, Mo., U.S.A.), or can be prepared according to methods known inthe art (Methods in Carbohydrate Chemistry [Academic Press, 1963], Vol.2). Glycosidic radicals which are suitable for coupling to the acidicnucleus to provide suitable glycosides of formula (H) include, but arenot intended to be limited to, radicals of sugars such asβ-D-galactopyranose, α-D-galactopyranose, β-D-glucopyranose,α-D-glucopyranose, α-D-mannopyranose, N-acetylglucosamine, β-glucuronicacid and neuraminic acid. Other suitable glycosidic radicals includeradicals of oligosaccharide chains which by saccharide-chain splittingenzymes can be broken down to the level of a mono- or oligosaccharide,which in its turn can be directly split off from the dye nucleus withthe corresponding glycosidase. It is to be understood that sucholigosaccharide chains are chains consisting of 2 to 20, preferably 2 to7 monosaccharide units, such as maltopentose, maltohexose ormaltoheptose. The acidic nucleus is reacted with a mono- oroligosaccharide or a 1-halo-derivative thereof, where all hydroxylgroups are substituted with a protecting group according to methodsknown in the art of carbohydrate chemistry, to give per-O-substitutedglycosides, from which the glycosides of the acidic nucleus are obtainedby cleaving the protective groups according to methods known in the art.

The compounds of the general formula (H) where Y=H are reacted with theper-O-substituted 1-halosaccharides, preferably in the presence ofproton acceptors such as alkali hydroxides or alkali carbonates, inaqueous acetone or (under phase transfer conditions) in awater/chloroform or water/benzene mixture. This procedure canfurthermore be carried out by first converting the acidic nucleus withalkali hydroxide or alcoholate into alkali salts or, using possiblysubstituted amines, into ammonium salts, and then reacting these withthe per-O-substituted 1-halosaccharides in dipolar aprotic solvents suchas acetone, dimethylsulfoxide, dichloromethane, tetrahydrofuran ordimethylformamide. Furthermore in the synthesis of per-O-substitutedglycosides from acidic nuclei and per-O-substituted 1-halosaccharides,additives in the form of single silver salts or mixtures of silversalts, such as silver oxide, silver carbonate, silver carbonate onCelite (Johns-Manville Corp., Denver, Colo., U.S.A.), silver triflate orsilver salicylate, and/or of single mercury salts or mixtures of mercurysalts, such as mercury bromide, mercury cyanide, mercury acetate ormercury oxide, and/or of single cadmium salts or mixtures of cadmiumsalts such as cadmium carbonate or cadmium oxide, possibly with the useof drying agents such as calcium chloride, a molecular seive or Drierite(W. A. Hammond Drierite Co., Xenia, Ohio, U.S.A.), in solvents such asmethylene chloride, chloroform, benzene, toluene, ethyl acetate,quinoline, tetrahydrofuran or dioxane have proven effective. In thesynthesis of α-linked glycosides, an acidic nucleus of the generalformula (H) where Y=H is melted with a saccharide whose hydroxy groupsare substituted with a protective group, preferably an acetyl-group, inthe presence of a Lewis acid, such as zinc chloride (see Chem. Ber. 66,378-383 [1933] and Methods in Carbohydrate Chemistry, Academic Press,1967, Vol. 2, pp. 345-347). The temperature of the reaction ispreferably between 80° and 130° C., more preferably between 110° and130° C.

Removing the protecting groups from the per-O-substituted glycosides toform glycosides of general formula (H) is performed according to methodsknown in the art of carbohydrate chemistry (see Advances in CarbohydrateChem. 12, 157 (1976), such as with the protective acyl-groups withsodium methylate, barium methylate or ammonia in methanol. Especiallysuitable as a protecting group commonly used in carbohydrate chemistryis an acetyl, benzoyl, benzyl or trimethylsilyl-radical.

Acidic nuclei of the general formula (H) where Y is the radical of anoligosaccharide chain of from about 2 to 20 monosaccharide unitsattached via α-1-4 glucosidic linkages can additionally be prepared fromthe α- and β- glucosides by an enzymatic process first described byFrench, et al., J. Am. Chem. Soc. 76, 2387 (1954), and later byWallenfels, et al., Carbohydrate Research 61, 359 (1978), involving thetransfer of the glucoside to a preformed polysaccharide chain by theenzyme (1-4)-α-glucan-4-glucosyltransferase (also known ascyclomaltodextrin glucanotransferase; EC 2.4.1.19).

Esters of merocyanine dyes of the general formula (A) are useful aschromogenic esterase and protease substrates. Such esters can beprepared by methods known in the art of organic chemistry by firstreacting known derivatives of carboxylic acids with a suitable acidicnucleus to provide a reactive electrophilic acid nucleus derivative ofthe formula (H) where Y is ##STR38## where V is alkyl, substituted alkyl(particularly aminoalkyl) or aryl. This derivative is then condensedwith an active methyl quaternary salt of the formula (K) (basic nucleus)to afford chromogenic merocyanine enzyme substrates.

Such known derivatives of carboxylic acids of the formula Y-OH include,but are not intended to be limited to, amino acid residues, preferablyresidues of naturally occurring α-amino acids in their L- or D- form oralso in their racemic form, the residues of glycine, alanine, valine,leucine, isoleucine, phenylalanine and tyrosine being preferred, the L-forms thereof being more preferred. Any free hydroxyl groups possiblypresent may be acylated and preferably acetylated. The peptide residuesin this definition of Y-OH are to be understood to be, for example,amino acids or peptides from between about 2 to 5 amino acid units suchas di-, tri-, tetra-, and pentapeptides, di- and tripeptides beingpreferred, the amino acid components thereof being the above-mentionedamino acids. It is also to be understood that the amino groups of suchamino acids or peptides may be protected with nitrogen protecting groupsknown in the art of peptide chemistry (see T. W. Green, ProtectiveGroups in Organic synthesis, J. Wiley and Sons, New York, N.Y., 1981,pp. 218-287) including, for example, acyl, oxycarbonyl, thiocarbonyl,sulphonyl, especially p-toluenesulphonyl (Tosyl, Ts), sulphenyl, vinyl,cyclohexenyl, and carbamoyl, especially t-butyl-(BOC) and benzyl-(CBz)carbamoyl radicals. Such esters may also be similarly prepared byreacting a compound of the general formula (H) where Y=H with acarboxylic acid, amino acid or peptide, Y-OH as defined above, or withan appropriate reactive derivative thereof, employing methods known inthe art of organic chemistry (see J. March, Advanced Organic Chemistry:Reactions, Mechanism and Structure, McGraw-Hill Book Co., New York, NH,1968, pp. 319-323). The reactive derivatives used can be, for example,acid chlorides or bromides, or mixed anhydrides conventionally used inpeptide synthesis, such as those with ethyl chloroformate, or activeesters such as those of N-hydroxysuccinimide.

Similarly, inorganic esters can be prepared according to methods knownin the art of organic systhesis. The known derivatives of inorganicacids Y-OH, such as phosphoric acid, e.g., compound where ##STR39## orsulfuric acid where ##STR40## are reacted with a compound of the generalformula (H) where Y=H employing methods known in the art of organicchemistry, such as shown in Koller and Wolfbeis, Monatsh. 116, 65 (1985)for inorganic esters of certain coumarins.

ANALYTICAL METHODS

The chromogenic enzyme substrate compounds of the present invention areuseful in analytical test systems which require the measurement of theamount of enzyme present therein, particularly those analytical testsystems employing enzyme-labeled assay reagents. Such analytical testsystems include, but are not intended to be limited to, enzymeimmunoassays known in the art as competitive, sandwich and immunometrictechniques where the amount of enzyme label in a particular fractionthereof can be measured and correlated to the amount of analyte underdetermination obtained from a liquid test sample.

The use of specific binding substances, such as antigens, haptens,antibodies, lectins, receptors, avidin, and other binding proteins, andpolynucleotides, labeled with an enzyme have been recently developed andapplied to the measurement of substances in biological fluids (see, forexample, Clin. Chem., Vol. 22, p. 1232 (1976); Reissue U.S. Pat. No.31,006; and U.K. Patent No. 2,019,308). Generally, such measurementdepends upon the ability of a binding substance, e.g., an antibody or anantigen, to bind to a specific analyte wherein a labeled reagentcomprising such binding substance labeled with an enzyme is employed todetermine the extent of such binding. Typically, the extent of bindingis determined by measuring the amount of enzyme labels present in thelabeled reagent which either has or has not participated in a bindingreaction with the analyte, wherein the amount of enzyme detected andmeasured can be correlated to the amount of analyte present in a liquidtest sample.

The chromogenic enzyme substrate compounds of the present invention areparticularly useful in analytical test systems as heretofore describedwhere an analytical test device comprising a carrier matrix incorporatedwith the chromogenic enzyme substrate compound of the present inventionis employed, the nature of the enzyme-specific moiety thereof depending,of course, upon the particular enzyme being detected.

The nature of the material of such carrier matrix can be of anysubstance capable of being incorporated with the chromogenic enzymesubstrate compound of the present invention, such as those utilized forreagent strips for solution analysis. For example, U.S. Pat. No.3,846,247 describes the use of felt, porous ceramic strips, and woven ormatted glass fibers. As substitutes for paper, U.S. Pat. No. 3,552,928describes the use of wood sticks, cloth, sponge material, andargilaceous substances. The use of synthetic resin fleeces and glassfiber felts in place of paper is suggested in British Pat. No. 1,39,139,and British Pat. No. 1,349,623 teaches the use of a light-permeablemeshwork of thin filaments as a cover for an underlying paper matrix.This reference also teaches impregnating the paper with part of areagent system and impregnating the meshwork with other potentiallyincompatible reagents. French Pat. No. 2,170,397 describes the use ofcarrier matrices having greater than 50% polyamide fibers therein.Another approach to carrier matrices is described in U.S. Pat. No.4,046,513 wherein the concept of printing reagents onto a suitablecarrier matrix is employed. U.S. Pat. No. 4,046,514 describes theinterweaving or knitting of filaments bearing reagents in a reactantsystem. All such carrier matrix concepts can be employed in the presentinvention, as can others. Preferably, the carrier matrix comprises abibulous material, such as filter paper, whereby a solution of thechromogenic enzyme substrate compound of the present invention isemployed to impregnate the matrix. It can also comprise a system whichphysically entraps the assay reagents, such as polymeric microcapsules,which then rupture upon contact with the test sample. It can comprise asystem wherein the assay reagents are homogeneously combined with thecarrier matrix in a fluid or semi-fluid state, which later hardens orsets, thereby entrapping the assay reagents.

In a preferred embodiment, the carrier matrix is a bibulous material inthe form of a zone or layer incorporated with the chromogenic enzymesubstrate compound of the present invention which is employed where aparticular assay is performed in a liquid environment employing aninsoluble assay reagent known in the art to physically separate the freespecies of the labeled reagent from the bound species of the labeledreagent. According to such assay system, an aliquot of liquid containingthe free species is removed and applied to the carrier matrix whereinthe chromogenic enzyme substrate compound incorporated therein interactswith the enzyme label of the labeled reagent of the free species fromthe liquid test sample to provide a detectable signal which can bevisibly observed and/or measured with an appropriate instrument, such asa spectrophotometer.

Similarly, a test device comprising two or more carrier matrices in theform of, for example, an uppermost layer or zone and a lowermost layeror zone can be employed. The lowermost layer of such test device can beincorporated with the chromogenic enzyme substrate compound of thepresent invention wherein a liquid test sample containing analyte underdetermination is applied to the uppermost layer of the device. Theanalyte which diffuses therein participates in the necessary bindingreactions to generate a free and bound (i.e., immobilized) species ofthe enzyme labeled reagent therein as described above. Accordingly, thefree species of the labeled reagent so generated is free to migrate intothe lowermost layer where the enzyme label of the free species cleavesthe enzymatically-cleavable group of the chromogenic enzyme substratecompound of the present invention incorporated therein to provide ameasurable, detectable signal as heretofore described.

The present invention will now be illustrated, but is not intended to belimited, by the following examples.

EXAMPLES

The synthesis of merocyanine substrates as described in the Examplesbelow involves two major steps. With reference to FIGS. 2 through 5 ofthe drawings, the first step is the synthesis of aβ-D-galactopyranosyloxyarylaldehyde from tetra-acetyl-protectedbromo-α-D-galactose and the corresponding hydroxyarylaldehyde in thepresence of silver (I) oxide and quinoline followed by deprotection ofthe hydroxyl group by alkaline hydrolysis with sodium methoxide. Thesecond step is the coupling of the substrate-modified arylaldehyde withan active methyl quaternary amine salt to give the correspondingmerocyanine substrate.

A. Preparation of Arylaldehyde Intermediates

4-(β-D-Galactopyranosyloxy)-benzaldehyde (4)--A solution of4-hydroxybenzaldehyde (1) (Aldrich Chemical Co., Inc., Milwaukee, Wis.,U.S.A.) (6.1 g; 50 mmol) in 1M NaOH (50 mL) was treated at ambienttemperature with a solution of acetobromo-α-D-galactose (Sigma ChemicalCo., St. Louis, Mo., U.S.A.) (10.28 g; 25 mmol) in acetone (200 mL). Thereaction mixture was stirred for 22 hours, then the acetone was removedunder reduced pressure and the aqueous residue was extracted thrice withCHCl₃ (80 mL each). The combined CHCl₃ extracts were washed thrice with1M NaOH (100 mL each), twice with H₂ O (200 mL each) and finally withbrine (150 mL). The solution was then dried over MgSO₄, filtered andevaporated to dryness in vacuo to afford4-(tetra-O-acetyl-β-D-galactopyranosyloxy)-benzaldehyde (9.33 g; 82%) asa yellow foam used without further purification. [Identical to thatprepared by H-R. Rackwitz, Carbohydrate Research, 88:223-32(1981)]

IR (KBr) cm⁻¹ : 1740, 1685, 1595, 1362, 1218, 1063

¹ H NMR (CDCl₃)δ: 2.03 (s, 3H), 2.04 (s, 3H), 2.10 (s, 3H), 2.20 (s,3H), 4.21 (br. s, 3H), 5.03-5.63 (m, 4H), 7.06-7.95 (AB, 4H), 9.93 (s,1H).

A solution of 4-(tetra-O-acetyl-β-D-galactopyranosyloxy)-benzaldehyde(9.33 g; 20.6 mmol) in absolute methanol (250 mL) was treated withsodium methoxide (90 mg) and allowed to stir for 2 hours at ambienttemperature. The reaction was then neutralized by addition of glacialacetic acid (about 0.2 mL) and evaporated to dryness under reducedpressure. The crude product was crystallized from hot EtOH (250 mL) togive (4) (4.45 g; 75.9%) as fine yellow needles with mp=189°-92° C.[mp=155-7° C., Z. Csuros et al., Acta. Chim. Acad. Sci. Hung., 42(3),263-7(1964); mp=177° C., F. Konishi et al., Agric. Biol. Chem., 47(7),1419(1983)] The mother liquor was worked for a second crop (0.46 g;7.8%).

IR (KBr) cm⁻¹ : 3350, 1685, 1600, 1515, 1250, 1090.

¹ H NMR (DMSO-d₆)δ: 3.40-3.85 (m, 6H), 4.65 (v.v.br. s, 4H), 5.01 (d,J=7Hz, 1H), 7.10-7.95 (AB, 4H), 9.89 (s, 1H);

¹³ C NMR (DMSO-d₆)ppm: 191.44, 162,38, 131.68, 130.64, 116.60, 100.60,75.82, 73.42, 70.42, 68.34, 60.60.

4-(β-D-Galactopyranosyloxy)-1-naphthaldehyde (5)--A solution of4-hydroxynaphthaldehyde (2) (Trans World Chemical Co., Rockville, Md.,U.S.A.) (4.304 g, 25 mmol) in aqueous 1.0M NaOH (25 mL) was treated witha solution of acetobromo-α-D-galactose (5.14 g, 12.5 mmol) in acetone(100 mL). The reaction mixture was stirred at ambient temperature for21.5 hours then the acetone was removed under reduced pressure. Theresulting dark mixture was extracted four times with CHCl₃ (40 mL each)then the combined CHCl₃ layers were washed thrice with aqueous 1.0M NaOH(50 mL each), twice with H₂ O (50 mL each) and once with brine (50 mL).The CHCl₃ solution was then dried over MgSO₄, filtered and evaporated todryness in vacuo to give crude product as a biege foam (4.02 g). Onecrystallization from EtOAc/hexane afforded4-(tetra-O-acetyl-β-D-galactopyranosyloxy)-1-naphthaldehyde (2.79 gm,44.4%) as analytically pure white rods with mp= 177°-8° C.

Analysis: Calculated for C₂₅ H₂₆ O₁₁ : C, 59.76; H, 5.22. Found: C,59.39; H, 5.25.

IR (KBr) cm⁻¹ : 1740, 1682, 1600, 1572, 1510, 1368, 1220, 1060, 770.

¹ H NMR (DMSO-d₆)δ: 2.00 (s, 3H); 2.03 (s, 3H); 2.05 (s, 3H); 2.18 (s,3H); 4.20 (d, J=6 Hz, 2H); 4.65 (t, J=6 Hz, 1H); 5.48 (br. s, 3H); 5.90(br. d, J=6 Hz, 1H); 7.37 (d, J=8 Hz, 2H); 7.55-7.90 (m, 2H); 8.00-8.35(m, 2H); 9.12-9.35 (m, 1H); 10.28 (s, 1H).

¹³ C NMR (DMSO-d₆)ppm: 192.68, 169.92, 169.79, 169.46, 156.72, 138.64,131.23, 129.60, 126.94, 126.02, 124.66, 124.27, 121.47, 107.56, 97.61,70.94, 69.90, 68.34, 67.24, 61.32, 20.29 (4 coincident bands).

A solution of4-(tetra-O-acetyl-β-D-galactopyranosyloxy)-1-naphthaldehyde (1.67 g;3.32 mmol) in HPLC-grade methanol (40 mL) was heated in a 60° C. bathand treated with sodium methoxide (15 mg). Within three minutes a thickwhite solid had separated. After 30 minutes, the reaction was cooled inice and the solid filtered, washed twice with ice-cold methanol andvacuum dried to give (5) (1.08 g; 97%) as an analytically pure fluffywhite solid with no mp<255° C.

Analysis: Calculated for C₁₇ H₁₈ O₇ : C, 61.07; H, 5.43. Found: C,61.10; H, 5.50.

IR (KBr) cm⁻¹ : 3400, 1665, 1574, 1513, 1254, 1223, 1100, 768.

¹ H NMR (DMSO-d₆)δ: 3.44-3.64 (m, 3H); 3.68-3.88 (m, 3H); 4.60 (d, J=4.6Hz, 1H); 4.69 (t, J=5.5 Hz, 1H); 4.96 (d, J=5.7 Hz, 1H); 5.19 (d, J=7.7Hz, 1H); 5.41 (d, J=5.4 Hz, 1H); 7.35 (d, J=8.2 Hz, 1H); 7.61-7.67 (m,1H); 7.72-7.78 (m, 1H); 8.14 (d, J=8.2 Hz, 1H); 8.44 (d, J=7.8 Hz, 1H);9.20 (d, J=8.1 Hz, 1H); 10.21 (s, 1H).

¹³ C NMR (DMSO-d₆)ppm: 192.59, 158.09, 139.14, 131.17, 129.30, 126.25,125.08, 125.03, 123.95, 122.68, 107.61, 101.00, 75.89, 73.16, 70.32,68.17, 60.42.

6-(β-D-Galactopyranosyloxy)-2-naphthaldehyde (6)--Under argon,6-hydroxy-2-naphthaldehyde (3) [R. Gandhi, J. Chem. Soc., 2530 (1955)](4.4 g, 25.6 mmole) was dissolved in 100 mL of quinoline to give a lightyellow solution. Then acetobromo-α-D-galactose (21.05 g, 51.2 mmole) andsilver (I) oxide (12.8 g, 55 mmole) were added and the resultingreaction mixture was stirred at room temperature under dark for 22hours. The reaction mixture was filtered and the filter-cake was washedwith EtOAc thoroughly. The dark reddish brown filtrate was then washedwith 1.25N HCl until the washing was very acidic. The acidic aqueoussolution was then extracted with EtOAc. The EtOAc solutions werecombined and washed with 5% NaHCO₃ and saturated NaCl solutions, driedover anhydrous MgSO₄, filtered and concentrated to give a brown viscousmaterial which was dissolved in CHCl₃ and flash-chromatographed with 500mL silica gel eluted with CH₂ Cl₂ /CH₃ OH (10/0.1, v/v) to give about 13g of 6-(tetra-O-acetyl-β-D-galactopyranosyloxy)-2-naphthaldehyde as anoff-white solid.

Under argon, 6-(tetra-O-acetyl-β-D-galactopyranosyloxy)-2-naphthaldehydeobtained above (12.8 g, 25.5 mmole) was dissolved in 100 mL of methanoland sodium methoxide (1 g, 18.5 mmole) was added. The resulting reactionmixture was heated in a 60° C. oil bath for one-half hour. The reactionmixture was allowed to be adsorbed onto 60 mL of silica gel while beingconcentrated and then flash-chromatographed with 800 mL of silica geleluted with CH₂ Cl₂ /CH₃ OH (8.5/1.5, v/v) to give an off-white solid.Recrystallization from absolute ethanol yielded 6.7 g (78.8%) of a whitesolid (6) mp=193° C. (dec.)

Analysis: Calculated for C₁₇ H₁₈ O₇.1/10 H₂ O: C, 60.75; H, 5.46. Found:C, 60.60; H, 5.63.

IR (KBr) cm⁻¹ : 3403, 1681, 1624, 1477, 1267, 1182, 1071, 781.

¹ H NMR (DMSO-d₆)δ: 3.42-3.59 (m, 3H); 3.61-3.78 (m, 3H); 4.55 (d, J=5,1H); 4.68 (t, J=5, 1H); 4.90 (d, J=5, 1H); 7.38 (dd, J=9, J=2, 1H); 7.57(d, J=2, 1H); 7.85 (d, J=8.6, 1H); 7.92 (d, J=8.6, 1H); 8.11 (d, J=9,1H); 8.51 (s, 1H); 10.09 (s, 1H).

¹³ C NMR (DMSO-d₆)ppm: 60.39, 68.16, 70.31, 73,37, 75.69, 100.91,110.61, 119.90, 122,92, 127.91, 128.03, 131.20, 132.34, 134.18, 137.41,157,84, 192.45.

B. Preparation of Substrate Compounds by Convergent Synthesis

1-Ethyl-2-(4'-β-D-galactopyranosyloxystyryl)-3, 3-dimethylindoleniumiodide (16)--A mixture of 4-(β-D-galactopyranosyloxy)-benzaldehyde (4)(1 g, 3.5 mmole), 2,3,3-trimethylindolenine ethiodide (7) [H. Richter &R. L. Dresser, J. Chem. Eng. Data, 9(3), 406-7 (1964)] (1.1 g, 3.5mmole) and 20 mL of anhydrous pyridine was heated in a 65° C. oil bathto give a dark orange solution. After four hours of reaction, yellowsolid separated out. The pyridine was evaporated off under reducedpressure. The solid was dissolved in hot methanol, adsorbed onto 10 mLof silica gel while concentrating the solution, thenflash-chromatographed with 300 mL of silica gel eluted with CH₂ Cl₂ /CH₃OH (8.5/1.5, v/v) to give 800 mg (40%) of substrate (16) mp 139° C.(dec.)

IR (KBr) cm⁻¹ : 3372, 1588, 1525, 1480, 1246, 1174, 1071, 768.

¹ H NMR (DMSO-d₆)δ: 1.45 (t, J=5, 3H); 1.8 (s, 6H); 3.38-3.60 (m, 3H);3.65-3.74 (m, 3H); 4.56 (d, J=4.6, 1H); 4.67 (m, 3H); 4.92 (d, J=5.7,1H); 7.21 (d, J=9, 2H); 7.60 (m, 3H); 7.89 (m, 2H); 8.24 (d, J=9, 2H);8.44 (d, J=16, 1H).

¹³ C NMR (DMSO-d₆)ppm: 13.69, 25.78, 52.11, 60.39, 68.14, 70.20, 73.29,75.77, 100.29, 110.27, 114.90, 116.79, 123.10, 128.19, 129.14, 133.05,140.42, 143.53, 143.78, 153.86, 161.85, 181.28.

MS(FAB, glycerol/methanol), m/z (rel int): 454 (M⁺, 21.9%); 292 (M⁺--162, 100%).

1-Ethyl-2-(4'-β-D-galactopyranosyloxynaphthyl-1-vinylene)-3,3-dimethylindoliniumiodide (17)--A stirred mixture of4-(β-D-galactopyranosyloxy)-1-naphthaldehyde (5) (5.95 g; 17.8 mmol) andmolecular seive 4 Å (15 g) in anhydrous pyridine (105 mL) was maintainedat 65°-8° C. under an inert gas atmosphere. This was treated every 45minutes for 2.25 hours with 1.68 g portions of2,3,3-trimethylindoleninium ethiodide (7) (H. Richter & R. L. Dresser,supra) (6.72 g total; 21.3 mmol) then allowed to stir for 2 hours. Thereaction was cooled, filtered and evaporated to dryness in vacuo. Theresidue was chromatographed on silica gel (475 g) developed withdichloromethane/methanol (85:15, v/v) solvent and fractions containing(18) were pooled and evaporated to dryness in vacuo. The crude productwas dissolved at ambient temperature in methanol (100 mL), diluted withethyl acetate (700 mL) and cooled at 0° C. overnight. The solid whichseparated was collected by filtration, washed with ethyl acetate andvacuum dried to give (17) (3.15 g). One recrystallization, as above,from methanol (75 mL) and ethyl acetate (600 mL) afforded analyticallypure (17) (2.65 g; 24%) as a red solid with mp=165° C. (dec.).

IR (KBr) cm⁻¹ : 3320, 1562, 1509, 1268, 1225, 1078, 762.

¹ HMR (DMF-d₇)δ: 1.62 (t, J=7, 3H); 2.00 (s, 6H); 3.47 (v br. s, 4H);3.56-3.84 (m, 3H); 3,87-4.12 (m, 3H); 4.98 (q, J=7, 2H); 5.37 (d, J=7.7,1H); 7.52 (d, J=8.5, 1H); 7.71 (m, 3H); 7.84 (t, J=7Hz, 1H); 7.95-8.12(m, 3H); 8.56 (t, J=7, 2H); 8.75 (d, J-8.5, 1H); 9.21 (d, J=16. 1H).

¹³ C NMR (DMF-d₇)ppm: 13.99, 26.73, 43.18, 53.14, 61.59, 69.23, 71.67,74.54, 77.03, 102.17, 109.80, 112.76, 115.64, 123.45, 123.76, 123.96,125.07, 126.81, 129.47, 129.86, 129.91, 131.26, 133.62, 141.48, 144.66,150.10, 159.37, 181.8.

    ______________________________________                                        MS (FAB, glycerol/                                                                             504 (M.sup.+, 4.5%) 342 (M-162,                              methanol) m/z    100%) 127 (I.sup.+, 95%).                                    (rel int):                                                                    ______________________________________                                    

Analysis: calculated for C₃₀ H₃₄ INO₆.H₂ O: C, 55.47; H, 5.59; N, 2.16.Found: C, 55.31; H, 5.55; N, 2.02.

1-Ethyl-2-(6'-β-D-galactopyranosyloxynaphthyl-2'-vinylene)-3,3-dimethylindoleniumiodide (18)--Under argon, a mixture of6-(β-D-galactopyranosyloxy)-2-naphthaldehyde, (6) (2.23 g, 6.7 mmole),2,3,3-trimethylindolenine ethiodide (7) (H. Richter & R. L. Dresser,supra) (2.1 g, 6.7 mmole) and 40 mL of anhydrous pyridine was heated ina 70° C. oil bath for 21 hours. Then pyridine was evaporated off underreduced pressure to give a viscous dark reddish brown residue which wasdissolved in CH₂ Cl₂ /CH₃ OH (9/1, v/v) and flash-chromatographed with450 mL silica gel and eluted with CH₂ Cl₂ /CH₃ OH (9:1, v/v) to give 2 g(48%) of substrate (18) as a red solid. mp 177° C. (dec.)

IR(KBr) cm⁻¹ : 3400, 1587, 1470, 1310, 1189, 1072, 760.

¹ H NMR (DMSO-d₆)δ: 1.48 (t, J=5, 3H); 1.85 (s, 6H); 3.43-3.59 (m, 3H);3.61-3.77 (m, 3H); 4.57 (d, J=4.5, 1H); 4.69 (t, J=5.5, 1H); 4.76 (q,J=7, 2H); 4.92 (d, J=5.7, 1H); 5.08 (d, J=7.7, 1H); 5.26 (d, J=5.1, 1H);7.38 (dd, J=9, J=2.4, 1H); 7.58 (d, J=2.2, 1H); 7.64 (m, 2H); 7.76 (d,J=16, 1H), 7.89-8.03 (m, 4H), 8.37 (dd, J=9, J=1.4, 1H); 8.61 (d, J=16,1H); 8.73 (s, 1H).

¹³ C NMR (DMSO-d₆) ppm: 13.35, 25.49, 42.00, 52.09, 60,30, 68.02, 70.24,73.27, 75.57, 100.93, 110.84, 111.29, 114.77, 119.85, 122,76, 124.68,127.71, 128.37, 128.86, 129.12, 130.12, 130.79, 134.04, 136.57, 140.15,143.64, 154.04, 157.85, 181.33.

MS(FAB, glycerol) m/z (rel int): 504 (M⁺, 10%); 342 (M-162, 45%).

MS (EI) m/z: 127 (I⁺, 90%).

3-Ethyl-2-(4'-β-D-galactopyranosyloxystyryl) benzothiazolium chloride(19)--A solution of (4) (0.284 g; 1 mmol) and 2-methylbenzothiazoleethiodide (8) (H. Richter & R. L. Dresser, supra) (0.336 g; 1.1 mmol) inanhydrous pyridine (5 mL) was heated in a 75° C. bath for about 20hours, then cooled to ambient temperature and evaporated to drynessunder reduced pressure. The dark purple residue was diluted with aminimal volume of methanol and loaded onto a 11/2" diameter×11" long lowpressure C-18 reverse phase column (Bondapak Prep C-18 packing, WatersDivision, Millipore Corp., Milford, Mass., U.S.A.) previouslyequilibrated and developed with 0.75M NaCl/MeOH (7:3, v/v). Fractionscontaining the yellow product were identified by incubating aliquotswith β-galactosidase in pH=8 phosphate buffer. These were pooled,evaporated to dryness in vacuo and de-salted by repeated methanolextractions leaving a yellow solid (0.47 g). This was further purifiedby twice passing the material through a 11/8"×33" Sephadex LH-20 column(Pharmacia LKB Biotechnology Inc., Piscatway, N.J., U.S.A.) packed anddeveloped with methanol. As before, fractions containing the desiredproduct were identified by incubating aliquots with β-galactosidase inpH=8 phosphate buffer then pooled and evaporated to dryness in vacuo atambient temperature for two days to afford (19) (0.14 g; 29%) as anorange solid. HPLC analysis on a single Waters μ-bondapak C-18 column(Waters Division, Millipore Corp., Milford, Mass., U.S.A.) using CH₃CN/0.01M NaH₂ PO₄ (1:4, v/v) solvent flowing at 1.0 mL/minute and either254 nm or 410 nm detection revealed only one band with t_(R) =9.9minutes.

IR (KBr) cm⁻¹ : 3360, 1595, 1510, 1227, 1180, 1070.

¹ H NMR (DMSO-d₆)δ: 1.45 (br. t, J=8 Hz, 3H), 3.10-3.85 (m, 7H),4.55-5.30 (m, 6H), 7.18 (d, J=8 Hz, 2H), 7.65-8.55 (m, 8H).

¹³ C NMR (DMSO-d₆) ppm: 171.61, 168.16, 160.88, 145.11, 140.79, 132.01,129,41, 128.11, 127.65, 124.40, 116.66, 116.42, 111.20, 100.40, 75.69,73.41, 70.29, 67.85, 60.21, 44.14, 14.11.

3-Ethyl-2-(4'-β-D-galactopyranosyloxynaphthyl-1'-vinylene)-benzothiazoliumiodide (20)--Approximately equal amounts of 4-(β-D-galactopyranosyloxy)-1-naphthaldehyde (5) and 2-methylbenzothiazole ethiodide (8) (H.Richter & R. L. Dresser, supra) were heated at reflux for about 1 minutein ethanol containing a small amount of piperidine. The initiallycolorless mixture became red-orange during this time. The presence of(20) in the reaction mixture was determined by mixing a portion of thereaction mixture with an equal amount of dimethylformamide, dilutingthis with 0.1M phosphate buffer pH=7.0, and then treating the resultingsolution with β-galactosidase. When the enzyme was added the solutionturned from yellow in color to violet.

3-Ethyl-2-(4'-β-D-galactopyranosyloxynaphthyl-1'-vinylene)-6-methoxybenzothiazoliumiodide (21)--Under argon, 6-methoxy-2-methylbenzothiazole (5 g, 27.9mmole, Aldrich Chemical Company, Inc., Milwaukee, Wis., U.S.A.) andethyliodide (5.4 mL, 67 mmole, Aldrich Chemical Company, Inc.,Milwaukee, Wis., U.S.A.) were mixed and heated in a 60° C. oil bath forabout 20 hours. The solid separated out was filtered, washed thoroughlywith acetone and dried to give a white solid of6-methoxy-2-methylbenzothiazole ethiodide (9) (2 g, 21%), mp=180°-182°C.

IR (KBr) cm⁻¹ : 2968, 1601, 1481, 1443, 1251, 1048, 853, 814.

¹ H NMR (DMSO-d₆)δ: 1.50 (t, J=7Hz, 3H), 3.25 (s, 3H), 3.95 (s, 3H) 4,80(q, J=7 Hz 2H), 7.40-8.50 (m, 3H).

MS (FAB, glycerol/methanol) m/z (rel int); 308 (M⁺, 100%).

Analysis: calculated for C₁₁ H₁₄ INOS: C, 39.42; H, 4.21; N, 4.18.Found: C, 39.62; H, 4.21; N, 4.34.

Under argon, a mixture of 4-(β-D-galactopyranosyloxy)naphthaldehyde (5)(0.5 g, 1.5 mmole), 6-methoxy-2-methylbenzothiazole ethiodide (9) (0.75g, 2.3 mmole) and 10 mL of anhydrous pyridine is heated at 65° C. oilbath. Orange solid separated out from the reaction mixture gradually.After five hours of reaction, the reaction mixture was cooled to roomtemperature, filtered, and the solid washed with pyridine, CHCl₃ and CH₃OH to give a bright orange solid. After drying under reduced pressure atroom temperature overnight yielded 0.5 g (50%) of substrate (21), mp229° C. (dec.).

Analysis: Calculated for C₂₈ H₃₀ INO₇ S: C, 51.62; H, 4.64; N, 2.15.Found: C, 51.38; H, 4.53; N, 2.30.

IR (KBr)cm⁻¹ : 3380, 1603, 1566, 1253, 1227, 1079, 770.

¹ H NMR (DMSO-d₆)δ: 1.48 (t, J=7, 3H); 3.48-3.64 (m, 3H); 3.71-3.87 (m,3H); 3.95 (s, 3H); 4.62 (d, J=5, 1H); 4.70 (t, J=5, 1H); 4.88-5.02 (m,3H); 5.18 (d, J=7.6, 1H); 5.44 (d, J=5, 1H); 7.37 (d, J=8.5, 1H); 7.46(dd, J=7.1, J=2.2, 1H); 7.65 (t, J=7.6, 1H); 7.76 (t, J=7.0, 1H); 8.02(m, 2H); 8.21 (d, J=9.3, 1H); 8.47 (m, 3H); 8.78 (d, J=15.5, 1H).

¹³ C NMR (DMSO-d₆)ppm: 13.36, 43.68, 55.51, 59.68, 67.38, 69.48, 72.30,75.11, 100.26, 105.95, 107.97, 112.01, 116.63, 117.75, 122.11, 123.04,124.15, 125.08, 127.30, 128.12, 129.26, 131.19, 134.09, 142.77, 156.29,158.51, 167.59.

MS(FAB, glycerol/methanol/HCl) m/z (rel int): 362-(M-162, 12%).

MS(EI) m/z (rel int): 127 (I⁺, 100%).

2-(4'-β-D-galactopyranosyloxynaphthyl-1'-vinylene)-3-methyl-β-naphthothiazoliumiodide (22)--Under argon, a mixture of4-(β-D-galactopyranosyloxy)naphthaldehyde (5) (0.50 g, 1.5 mmole),2-methyl-β-naphthothiazole methiodide (10) (H. Richter & R. L. Dresser,supra) (0.61 g, 1.8 mmole), 20 mL of anhydrous pyridine and 10 mL ofanhydrous DMF was heated in a 65° C. oil bath for five hours to give adark brownish red mixture. Thin-layer chromatography (TLC) showed thepresence of large amounts of aldehyde starting material. Another portionof 2-methyl-β-naphthothiazole methiodide (10) (0.61 g, 1.8 mmole) wasadded to the reaction mixture. After an additional two hours ofreaction, the reaction mixture was filtered and pyridine and DMF wereevaporated off under reduced pressure. About 1 mL of KI in methanolsolution (1 g KI/8 mL methanol) was added to the residue and the mixturewas then adsorbed onto 10 mL of silica gel while the methanol was beingevaporated off under reduced pressure. Flash-chromatography with 200 mLof silica gel eluted with CH₂ Cl₂ /CH₃ OH (8.5/1.5, v/v) followed byrecrystallization from CH₃ OH/Et₂ O yielded 27 mg (3%) of substrate (22)as a red solid, mp 160° C.

IR (KBr) cm⁻¹ : 3440, 1620, 1564, 1230, 1076, 775.

¹ H NMR (DMSO-d₆)δ: 3.40-3.68 (m, 3H); 3.72-3.90 (m, 3H); 4.63 (d, J=5,1H); 4.72 (t, J=5, 1H); 4.82 (s, 3H); 4.98 (d, J=5, 1H); 5.19 (d, J=7.7,1H); 5.44 (d, J=5, 1H); 7.38 (d, J=8.6, 1H); 7.64 (m, 1H); 7.76 (m, 1H);7.89 (m, 2H); 8.15 (m, 1H); 8.33 (d, J=9, 2H); 8.43 (m, 2H); 8.52 (d,J=8.6, 2H); 8.83 (d, J=15, 1H); 9.00 (d, J=7.7, 1H).

¹³ C NMR (DMSO-d₆)ppm: 42.0, 60.36, 68.18, 70.37, 73.16, 75.87, 100.91,107.57, 108.85, 113.79, 119.63, 122.75, 122.93, 124.04, 124.95, 125.91,127.85, 128.09, 128.52, 128.73, 129.92, 131.13, 132.08, 133.58, 137.85,139,28, 143.15, 145.41, 156.53, 170.15.

3-Ethyl-2-(4'-β-D-galactopyranosyloxynaphthyl-1'-vinylene)-5-methylbenzothiazoliumiodide (23) Under argon, 2,5-dimethylbenzothiazole (10 g, 61 mmole,Aldrich Chemical Company, Inc., Milwaukee, Wis.) and ethyliodide (9.8mL, 123 mmole, Aldrich Chemical Company, Inc., Milwaukee, Wis.) weremixed and heated in a 65° C. oil bath for about 23 hours. The solidseparated out was filtered, washed thoroughly with acetone,recrystallized from absolute ethanol and dried to give a white solid of2,5-dimethylbenzothiazole ethiodide (11) (3 g, 15.4%), mp-202°-3° C.

Analysis: calculated for C₁₁ H₁₄ INS: C, 41.39; H, 4.42; N, 4.39. Found:C, 41.71; H, 4.44; N, 4.50.

A mixture of 4-(β-D-galactopyranosyloxy)-naphthaldehyde (5) (0.33 g, 1mmole), 2,5-dimethylbenzothiazole ethiodide (11) (0.38 g, 1.2 mmole), 3mL of pyridine and some molecular sieves was heated in a 120° C. oilbath in a closed flask for 7 minutes. Then the reaction mixture wascooled and 30 ml of acetone was added. The red solid separated out wasfiltered and washed with acetone. The solid was then dissolved inpyridine and purified with silica gel column eluted with CH₂ Cl₂ /CH₃ OH(3/7, v/v) to give 0.2 g of substrate (23) as a red solid, mp 160° C.(dec.).

MS(FAB, diethiothreitol/dithioerythritol/methanol) m/z: 508 (M⁺, 2.3%)346 (M-162, 30.1%).

3-Ethyl-2-(6'-β-D-galactopyranosyloxynaphthyl-2'-vinylene)-benzoselenazoliumiodide (24)--A mixture of 6-(β-D-galactopyranosyloxy)-2-naphthaldehyde(6) (34 mg, 0.1 mmole), 2-methylbenzoselenazole ethiodide (12) (H.Richter & R. L. Dresser, supra) (35 mg, 0.1 mmole) and 0.5 mL ofanhydrous pyridine was heated in a 70° C. oil bath for 20 hours. TLCanalysis with solvent CH₂ Cl₂ /CH₃ OH (8.5/1.5, v/v) showed the presenceof a yellow product spot which was extracted with 0.3M bicene buffer (pH7.9). On addition of β-galactosidase and a drop of 1N NaOH, the solutionturned to purple immediately.

3-(2"-Carboxyethyl)-2-(4'-β-D-galactopyranosyloxynaphthyl-1'-vinylene)-5-methylbenzoxazolium bromide, (25)--Under argon, a mixture of4-(β-D-galactopyranosyloxy)-naphthaldehyde (5) (0.5 g, 1.5 mmole),3-(2'-carboxyethyl)-2,5-dimethylbenzoxazolium bromide (13) (AldrichChemical Company, Inc.) (1.35 g, 4.5 mmole) and 10 mL of anhydrouspyridine was heated in a 65° C. oil bath for 31/2 hours. The reactionmixture was then cooled to room temperature and thenflash-chromatographed with 80 mL of silica gel eluted with CH₂ Cl₂ /CH₃OH (8.5/1.5, v/v) to give 240 mg (30%) of substrate (25) as a red solid,mp 150° C. (dec.).

IR (KBr) cm⁻¹ : 3219, 1730, 1606, 1567, 1512, 1270, 1224, 1077, 770.

¹³ C NMR (DMSO-d₆)δ: 20.0, 32.5, 44.07, 60.41, 68.16, 70.41, 73.26,75.73, 101.49, 108.87, 116.50, 122.80, 124.02, 125.27, 125.49, 127.24,127.73, 128.14, 129.77, 131.58, 136.79, 137.21, 139.38, 142.06, 145.21,151.07, 154.20, 165.20, 172.04.

MS (FAB, glycerol/methanol/HCL) m/z 536 (M⁺, 4%), 374 (M-162, (rel int):20%).

1-Ethyl-2-(4'-β-D-galactopyranosyloxynaphthyl-1'-vinylene)-quinoliniumiodide (26)--A mixture of 4-(β-D-galactopyranosyloxy)-naphthaldehyde (5)(0.33 g, 1 mmole), quinaldine ethiodide (14) (H. Richter & R. L.Dresser, supra) (0.30 g, 1 mmole), some molecular sieve (4 Å, 8-12 mesh)and 5 mL of anhydrous pyridine in a stoppered round-bottomed flask washeated in a 120° C. oil bath for 15 minutes. Thin-layer chromatographyshowed the presence of large amounts of aldehyde starting material. Anadditional portion of quinaldine ethiodide (0.1 g, 0.3 mmole) was addedand the resulting reaction mixture was heated for an additional tenminutes to give a dark viscous mixture. Then the reaction mixture wascooled, acetone was added and the resulting slurry was filtered andwashed with large amount of acetone to give a bright orange solid. Theorange crude product was dissolved in warm DMSO and was thenflash-chromatographed with 90 mL of silica gel eluted with CH₂ Cl₂ /CH₃OH (9/1, v/v). Orange solid crystallized out from the fractionscontaining the product and was filtered and dried under reduced pressureat room temperature overnight to yield 26 mg (4%) of substrate (26) as abright orange solid. mp 236°-237° C. (dec.).

IR (KBr) cm⁻¹ : 3367, 1603, 1568, 1339, 1219, 1076, 760.

¹ H NMR (DMSO-d₆)δ: 1.60 (t, J=7, 3H); 3.46-3.67 (m, 3H); 3.70-3.90 (m,3H); 4.62 (d, J=4.5, 1H); 4.71 (t, J=5.1, 1H); 4.97 (d, J=5.6, 1H); 5.17(m, 3H); 5.43 (d, J=5.2, 1H); 7.37 (d, J=8.5, 1H); 7.63 (t, J=7.5, 1H);7.73 (t, J=7.5, 1H); 7.86 (d, J=15.5, 1H); 7.94 (t, J=7.3, 1H); 8.19 (t,J=7.8, 1H); 8.33-8.51 (m, 3H); 8.57 (d, J=9.0, 1H); 8.63 (d, J=8.6, 1H);8.90 (d, J=9.0, 1H); 9.00 (d, J=15.2, 1H); 9.06 (d, J=9, 1H).

¹³ C NMR (DMSO-d₆)ppm: 12.51, 47.15, 61.91, 70.12, 72.37, 75.36, 78.32,105.22, 113.35, 123.40, 124.04, 127.10, 128.30, 128.86, 130.49, 130.63,131.52, 133.60, 133.79, 133.87, 134.68, 136.26, 138.33, 141.25, 144.45,150.34, 150.70, 162.70, 163.71.

    ______________________________________                                        MS (FAB, dithiothrietol/                                                                          488 (M.sup.+, 7.7%); 326                                  dithioerythritol/   (M-162, 100%)                                             methanol)m/z                                                                  (rel int):                                                                    MS(EI) m/z (rel int):                                                                             127 (I.sup.+, 30%).                                       ______________________________________                                    

1-Ethyl-2-(4'-β-D-galactopyranosyloxynaphthyl-1'-vinylene)-6-methoxyquinoliniumiodide (27)--Under argon, 6-methoxyquinaldine (10 g, 58 mmole, AldrichChemical Company, Inc., Milwaukee, Wis., U.S.A.) and ethyliodide (9.3mL, 116 mmole) were mixed and heated in a 65° C. oil bath for about 20hours. Acetone was added to the reaction mixture. The solid wasfiltered, washed thoroughly with acetone, recrystallized from absoluteethanol and dried to give a light yellow solid of 6-methoxyquinaldineethiodide (15) (4.2 g, 22%).

A mixture of 4-(β-D-galactopyranosyloxy)naphthaldehyde (5) (0.3 g, 0.9mmole), 6-methoxyquinaldine ethiodide (15) (0.36 g, 1.1 mmole), somemolecular sieve (3 Å, 8-12 mesh) and 2 ml of anhydrous pyridine washeated in a 120° C. oil bath for 10 minutes. DMF was added and theresulting reaction mixture was then flash-chromatographed with 90 mL ofsilica gel eluted with CH₂ Cl₂ /CH₃ OH (8/2, v/v) to give 26 mg of a redsolid of substrate (27) (6%). MS(FAB,dithiothreitol/dithioerythritol/methanol) m/z: 518 (M⁺, 7.1%); 356 (M⁺-162, 48.9%)

1-Ethyl-4-(4'-β-D-galactopyranosyloxynaphthyl-1'-vinylene)-quinoliniumiodide (29)--A mixture of 4-(β-D-galactopyranosyloxy)-naphthaldehyde (5)(34 mg, 0.1 mmole), lepidine ethiodide (28) (H. Richter & R. L. Dresser,supra) (30 mg, 0.1 mmole) and 0.5 mL of anhydrous pyridine was heated ina 70° C. oil bath for 3 hours. Then an additional of 60 mg of lepidineethiodide (28), was added and the reaction mixture was allowed to reactin the 70° C. oil bath for a total of 20 hours. TLC analysis withsolvent CH₂ Cl₂ /CH₃ OH (8/2, v/v) showed the presence of a yellowproduct spot which was extracted with 50 mM of phosphate buffer (pH7.4). On addition of β-galactosidase the yellow solution turned to bluecolor within 5 seconds.

C. Study of Chromogenic Substrate Properties

The properties of some of the compounds were studied and are reported inTable D.

D. Test Strip

The substrates at an indicated concentration in 0.3M Bicene buffer, pH7.9 and 4 mM MgCl₂ were impregnated into Whatman 54 paper (Whatman Inc.,Clifton, N.J., U.S.A.) and then air-dried for 1 hour at roomtemperature. A 0.5×1.0 cm pad of the paper was then mounted onto the endof a 0.5×8.125 cm polystyrene strip (Tricite®, Dow Chemical Co.,Midland, Mich., U.S.A.) previously laminated with a 2 mm strip of DoubleStick® double-faced adhesive tape (3M Company, St. Paul, Minn., U.S.A.).Thirty microliters of an indicated levels of β-galactosidase inphosphate buffer, pH 7.4, were then added to the substrate pad andreflectances were recorded at five second intervals at the opticalabsorption maximum of wavelength specified for the chromophore using theSERALYZER® reflectance meter (Miles Inc., Elkhart, Ind., U.S.A.).Reflectance values were linearized through a mathematical function andconverted to units identified as L(R) units.

The results were shown in FIGS. 6-8.

The present invention has been particularly described and exemplifiedabove. Obviously, many other variations and modifications of theinvention can be made without departing from the spirit and scopethereof.

                                      TABLE D                                     __________________________________________________________________________                                         Kcat                                     .sup.λ max   ε × 10.sup.3                                                                  Km mole/min per                             Compound                                                                            Substrate                                                                          Chromogen                                                                           Shift                                                                            Substrate                                                                          Chromogen                                                                           pKa                                                                              mM mole active site                         __________________________________________________________________________    16    406  522   116                                                                              25.3 45.0  7.3                                                                              0.018                                                                            1.43 × 10.sup.3                    17    456  580   124                                                                              28.0 82.0  6  0.09                                                                             7.8 × 10.sup.3                     18    430  530   100                                                                              32.2 --    8.3                                                                              0.08                                                                             1.3 × 10.sup.4                     19    393  492    99                                                                              --   43.0  7.6                                                                              0.017                                                                            3.04 × 10.sup.3                    20    yellow                                                                             violet                                                                              -- --   --    -- -- --                                       21    422  598   176                                                                              10.5 80.1  6.6                                                                              0.43                                                                             2.8 × 10.sup.3                     22    470  610   140                                                                              --   --    7  -- --                                       23    yellow                                                                             blue  -- --   --    -- -- --                                       24    yellow                                                                             purple                                                                              -- --   --    -- -- --                                       25    420  550   130                                                                              15.8 50.1  6.6                                                                              0.08                                                                             9.8 × 10.sup.2                     26    434  606   172                                                                              16.0 70.2  6.5                                                                              -- --                                       29    yellow                                                                             blue  -- --   --    -- -- --                                       __________________________________________________________________________

What is claimed is:
 1. A method for determining a particular enzyme in aliquid test sample, comprising the steps of:(a) contacting the testsample with a compound of the formula ##STR41## wherein Y is anenzymatically-cleavable group that is (i) capable of being cleaved fromsuch compound by said enzyme or (ii) capable of being modified by saidenzyme to produce a secondary substrate compound in which the modifiedenzymatically-cleavable group is cleavable from the compound by asecondary enzyme, in which case the secondary substrate compound iscontacted with said secondary enzyme; A represents a nonmetallic atomicgroup or residue which completes a 5- or 6-membered carbocyclic orheterocyclic ring or a fused ring system consisting of 5- and/or6-membered heterocyclic or carbocyclic rings; B represents a nonmetallicatomic group or residue which completes a 5- or 6-membered N-containingheterocyclic ring or a fused ring system consisting of 5- and/or6-membered heterocyclic or carbocyclic rings; R¹ is alkyl or aryl; R²and R³, which may be the same or different, are hydrogen or lower alkyl;m, n, and p, which may be the same or different, are integers from 0through 3 provided that m+n+p is at least 2; and X is a counterion(anion); and (b) measuring the resulting color generated by the cleavedmerocyanine indicator group.
 2. The method of claim 1 wherein saidenzymatically-cleavable group is a radical of a compound Y-OH selectedfrom the group consisting of sugars and derivatives thereof.
 3. Themethod of claim 2 wherein Y-OH is a sugar or derivative thereof selectedfrom the group consisting of α-D-glucose, α-D-galactose, α-D-glucose,β-D-glucose, α-D-mannose, N-acetylglucosamine and N-acetylneuraminicacid.
 4. The method of claim 2 wherein Y-OH is an oligosaccharide chainof from between about 2 to 20 monosaccharide units.
 5. The method ofclaim 2 wherein Y-OH is β-D-galactose, β-D-glucose or α-D-glucose. 6.The method of claim 1 wherein said enzyme-cleavable group is a radicalof a compound Y-OH selected from the group consisting of amino acids andpeptides.
 7. The method of claim 1 wherein said enzyme-cleavable groupis phosphate.
 8. The method of claim 1 wherein A represents substitutedor unsubstituted phenylene, naphthylene or anthrylene.
 9. The method ofclaim 8 wherein A represents 1,2-naphathylene, 1,4-phenylene,1,4-naphthylene, or 2,6-naphthylene.
 10. The method of claim 1 wherein Brepresents a nonmetallic atomic group or residue which completes a 5- or6-membered N-containing heterocyclic ring or a fused ring systemconsisting of three or less 5- and/or 6-membered heterocyclic orcarbocyclic rings.
 11. The method of claim 10 wherein said residuecompletes a ring or fused ring system selected from substituted orunsubstituted forms of indolenium, β-naphthothiazolium, benzoxazolium,benzothiazolium, quinolinium, thiazolium, benzoselenazolium, orbenzimidazolium.
 12. The method of claim 10 wherein said atomic group orresidue completes a ring or fused ring system of the formula: ##STR42##wherein Z is disubstituted methylene, vinylene, alkyl- oraryl-substituted N, O, S or Se, and wherein the phenyl ring in theformula is substituted or unsubstituted.
 13. The method of claim 1wherein R¹ is lower alkyl or phenyl.
 14. The method of claim 1 whereinR² and R³ are both hydrogen.
 15. The method of claim 1 wherein m+n+p is3, 4 or 5.